We have developed a simple and reliable assay to identify small molecule inhibitors of the Cch1-Mid1 Ca2+ channel. This complex promotes many critical aspects of fungal physiology. In Cryptococcus neoformans, Candida albicans and C. glabrata, Cch1-Mid1 signaling plays a crucial role in tolerating and surviving ER stress conditions that are induced by inhibitors of ergosterol biosynthesis, such as triazole antifungal drugs. C. neoformans is the leading cause of fungal meningitis in AIDS patients and other patients without adequate T-cell-dependent immune functions. Small molecule modulators would provide critical tools for studying Ca2+- mediated signaling events during ER stress and might even lead the way to the development of important adjuvants that might synergize with existing antifungals and enhance the treatment of life-threatening fungal infections. However, there are currently no inhibitors of Cch1 signaling. Our assay takes advantage of the model fungal pathogen C. neoformans, which requires the Cch1-Mid1 Ca2+ channel for its survival in low Ca2+ environments. On low Ca2+ media in the presence of small molecules that block Cch1 activity and prevent Cch1-mediated signaling, the growth of C. neoformans would be arrested. Thus the assay uses a simple readout (growth versus growth arrest) to identify small molecules that inhibit the Cch1-Mid1 channel. Because the assay is reproducible and has a simple readout, this assay is highly amenable to high-throughput screening. We will use a counter screen to prioritize the lead compounds and also validate the biological relevance of each molecule through patch clamp techniques and FRET- based Ca2+ imaging. We anticipate that a screen using this assay may provide a crucial step forward in understanding the role of the Cch1-Mid1 channel in critical Ca2+-mediated signaling events and in developing a novel strategy for treating fungal infections. This project is responsive to the program announcement for the development of assays for high-throughput drug screening. PUBLIC HEALTH RELEVANCE: This study aims to understand the role of a calcium channel that governs essential physiological processes in fungal pathogens as a means to assess its viability as a target for antifungal drug development. Because fungal infections are life threatening for patients with a suppressed immune response, the development of more efficacious and better-tolerated drugs is essential.